اثرتیمارهای مختلف سترون‌سازی و نوع جوانه بر رشد اولیه آناناس رقم md2 در شرایط درون شیشه‌ای

این پژوهش به منظور بررسی شرایط بهینه برای شروع کشت جوانه آناناس رقم  md2در شرایط درون شیشه ای با اجرای سه آزمایش مختلف انجام شد. در آزمایش اول، اثر تیمار سترون سازی بر آلودگی، مرگ و میر و میزان بقای ریزنمونه ها بررسی شد. هیپوکلریت سدیم و اتانول، به تنهایی یا در ترکیب، برای سترون سازی ریزنمونه ها در غلظت ها و مدت زمان های مختلف: هیپوکلریت سدیم دو و نیم درصد کلر فعال به مدت پنج، 10 و 15 دقیقه، اتانول 70 درصد به مدت سه، پنج و هفت دقیقه، هیپوکلریت سدیم یک درصد کلر فعال به مدت 10 دقیقه + اتانول 70 درصد به مدت سه دقیقه و هیپوکلریت سدیم دو و نیم درصد کلر فعال به مدت 15 دقیقه + اتانول 70 درصد یک دقیقه استفاده شد. در آزمایش دوم، جوانه‌ها بر اساس موقعیت آنها بر روی ساقه آناناس به پنج گروه: جوانه انتهایی، جوانه های قسمت اول، جوانه های قسمت دوم، جوانه های قسمت سوم، جوانه های بخش چهارم تقسیم شدند تا بیشترین میزان رشد جوانه مشخص شود. در آزمایش سوم، اثر غلظت های مختلف بنزیل آدنین شامل: صفر، 0/1، 0/5 و یک میلی گرم در لیتر به تنهایی یا همراه با 0/01میلی‌گرم در لیتر اسید نفتالین استیک بر رشد جوانه‌ها بررسی شد. کلیه کشت ها در دمای 2 ± 26 درجه سانتی گراد، شدت نور 3000 لوکس و دوره نوری 16 ساعت روشنایی قرار گرفتند. نتایج نشان داد که هیپوکلریت سدیم دو و نیم درصد کلر فعال به مدت 20 دقیقه بیشترین تاثیر را برای سترون سازی جوانه‌های آناناس داشت. کلیه غلظت های بنزیل آدنین مورد استفاده باعث رشد جوانه ها شد، ولی غلظت سه میلی گرم در لیتر بیشترین تاثیر را داشت. محیط فاقد تنظیم کننده های رشدکمترین میزان رشد جوانه را داشتند. جوانه های انتهایی و جوانه های جانبی بخش سوم به ترتیب با 93 درصد و 92 درصد بیشترین میزان رشد را داشتند.

effect of different disinfection treatments and bud type on growth initiation of pineapple cv. md2 in in vitro conditions

the aim of this study was to determine the optimal conditions for in vitro culture initiation of pineapple cv. md2 using tissue culture technique, in three different experiments. in the first experiment, the effect of sterilization treatment on contamination, mortality and development rate of explants was investigated. in the second experiment, the buds were divided into five groups based on their position on the pineapple stem to determine the highest rate of bud regeneration. in the third experiment, the effect of different concentrations of benzyl adenine (ba) (0, 0.1, 0.5 and 1 mgl-1) was studied alone or in combination with 0.01 mgl-1 of naphthalene acetic acid (naa) concentrations on regeneration of buds. the results showed that sodium hypochlorite (2.5%) in 20 minutes was the most effective for sterilizing pineapple buds. all concentrations of ba promoted bud regeneration but the 3 mgl-1 was the optimum. apical buds and the third section axillary buds had the highest regeneration rate at 93% and 92%, respectively. keywords: pineapple, benzyl adenine, apical bud, tissue culture, sodium hypochlorite. introductionpineapple [ananas comosus (l.) merr.] is one of the most economically important tropical fruit that belongs to bromeliaceae. pineapple plants can be propagated using vegetative materials of slips, suckers, stem sections or crowns. however, in all these three cases plant material is often limited, thus in vitro culture is a commercial alternative approach. in vitro micropropagation of pineapple plantlets has many advantages over conventional methods of vegetative propagation. tissue culture enhances rapid propagation of suckers to supplement conventional methods to produce pineapples to meet both local and international market demands (shamim et al., 2016). however, tissue culture initiation, the most critical step towards rapid in vitro propagation, is challenged by microbial contamination, exogenous growth hormone requirements and bud type for growth (vujovic et al., 2012). therefore, the aim of this study was to determine optimal sterilization protocols and ba concentration to initiate pineapple in vitro culture using apical and axillary buds as sources of explants. materials and methods in this study the buds of suckers of pineapple cv. md2 was used for in vitro culture in three different experiments. in the first experiment, explants were sterilized with sodium hypochlorite and ethanol at different concentrations, alone or in combination, and durations (2.5% sodium hypochlorite for 5, 10, 15 minutes, 70% ethanol for 3, 5, 7 minutes, 1% sodium hypochlorite for 10 minutes + 70% ethanol for 3 minutes and 2.5% sodium hypochlorite for 15 minutes + 70% ethanol for 1 minute), followed by three rinsing with sterilized distilled water under aseptic conditions. the explants were placed on solidified murashige and skoog (ms) based medium supplemented with 0.5 mgl-1 of benzyl adenine (ba) combined with 0.1 mgl-1 of naphthalene acetic acid (naa). in the second experiment, buds were grouped based on their position on the pineapple stem (apical, 1st section, 2nd section, 3rd section, and 4th section) to determine the highest rate of bud regeneration. following sterilization, different buds were transferred to ms medium containing 1 mgl-1 ba+ 0.1 mgl-1 naa at ph = 5.8. in the third experiment, the effect of different concentrations of ba (0, 0.1, 0.5 and 1 mgl-1) was studied alone or in combination with 0.01 mgl-1 naa concentrations on regeneration of buds. results and discussionthe results showed that sodium hypochlorite (2.5%) in 20 minutes was the most effective in sterilizing pineapple buds. the effectiveness of sodium hypochlorite (2.5%) enhanced with increasing exposure time. the highest explant regeneration rate was observed in the disinfection treatment with 2.5% sodium hypochlorite for 20 minutes (84.49%) followed by 2.5% sodium hypochlorite treatment for 15 minutes + 70% ethanol for 1 minute (75.39%). ethanol alone could not successfully disinfect pineapple buds. there was high level of harmful microbial contamination of plant tissues associated 70% ethanol treatment, hence the cultures deteriorated.the apical bud followed by the third section buds were the first to show growth and had the highest rate of regeneration (95% after two weeks). the third section buds recorded (90%) regeneration rate within 1-3 weeks.all concentrations of ba promoted bud regeneration. the highest regeneration rate was observed in the culture medium containing ba at concentration level of 3 mgl-1 and 0.1 mgl-1 naa with an average of 75.75%. the lowest regeneration rate in the ms culture medium without growth regulators with an average of 42%. these results are in agreement with findings of zuraida et al. (2011) and usman et al, (2013). referencesshamim, m., kumar, m., ranjan, t., kumar, r. r., pal, a. -k., kumar, v., jha, v. b. and kumar, p. 2016. importance of micropropagation in pineapple fordisease free plantlets and rapid multiplication. journal of pharmacognosy and phytochemistry, 5(4), pp. 359-362.usman, i. s. 2013. development of an efficient protocol for micro propagation of pineapple. african journal of agricultural research, 8(18), pp.2053-2056. doi: 10.5897/ajar12.1763vujovic, t., ruzic, d. j. and cerovic, r. 2012. in vitro shoot multiplication as influenced by repeated subculturing of shoots of contemporary fruit rootstocks. horticultural science, 39, pp.101-107. doi: 10.17221/208/2011-hortscizurida, a. r., shahanadz, a. h. n., harteeni, roowi, s., che radziah, c. m. z. and sreeramanan, s. 2011. a novel approach for rapid micropropagation of maspine pineapple shoots using liquid shake culture system. african journal of biotechnology, 10, pp.3859-3866.